Preparation of poly alpha-1,3-glucan esters and films therefrom

ABSTRACT

Poly alpha-1,3-glucan ester compounds are disclosed herein with a degree of substitution of about 0.05 to about 3.0. Also disclosed are methods of producing poly alpha-1,3-glucan ester compounds and films made therefrom.

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 14/136,168, filed Dec. 20, 2013 and claims the benefit of U.S.Provisional Application Nos. 61/746,328; 61/746,335 and 61/746,338; eachfiled Dec. 27, 2012, all of which are incorporated herein by referencein their entirety.

FIELD OF INVENTION

This invention is in the field of poly alpha-1,3-glucan derivatives.Specifically, this invention pertains to poly alpha-1,3-glucan esters,methods of their preparation and films made therefrom.

BACKGROUND

Driven by a desire to find new structural polysaccharides usingenzymatic syntheses or genetic engineering of microorganisms or planthosts, researchers have discovered polysaccharides that arebiodegradable, and that can be made economically from renewableresource-based feedstocks. One such polysaccharide is polyalpha-1,3-glucan, a glucan polymer characterized by havingalpha-1,3-glycosidic linkages. This polymer has been isolated bycontacting an aqueous solution of sucrose with a glucosyltransferaseenzyme isolated from Streptococcus salivarius (Simpson et al.,Microbiology 141:1451-1460, 1995). Films prepared from polyalpha-1,3-glucan tolerate temperatures up to 150° C. and provide anadvantage over polymers obtained from beta-1,4-linked polysaccharides(Ogawa et al., Fiber Differentiation Methods 47:353-362, 1980).

U.S. Pat. No. 7,000,000 disclosed the preparation of a polysaccharidefiber comprising hexose units, wherein at least 50% of the hexose unitswithin the polymer were linked via alpha-1,3-glycosidic linkages usingan S. salivarius gtfJ enzyme. This enzyme utilizes sucrose as asubstrate in a polymerization reaction producing poly alpha-1,3-glucanand fructose as end-products (Simpson et al., 1995). The disclosedpolymer formed a liquid crystalline solution when it was dissolved abovea critical concentration in a solvent or in a mixture comprising asolvent. From this solution, continuous, strong, cotton-like fibers,highly suitable for use in textiles, were spun and used.

Information regarding preparation of various derivatives of polyalpha-1,3-glucan and their application is sparse.

Yui et al. (Int. J. Biol. Macromol. 14:87-96, 1992) disclose using polyalpha-1,3-glucan extracted from the fruiting body of the fungus,Laetiporus silphureus, to synthesize poly alpha-1,3-glucan triacetate.The structure of this polymer was analyzed by X-ray crystallography.

Ogawa et al. (Carb. Poly. 3:287-297, 1983) used three different samplesof poly alpha-1,3-glucan to prepare poly alpha-1,3-glucan triacetate.One sample was isolated from a bacterial extracellular polysaccharide,and the other two samples were extracted from fruiting bodies of fungi.The structures of these polymers were analyzed by X-ray crystallography.

Development of new poly alpha-1,3-glucan ester derivatives and methodsof preparing such derivatives is desirable given their potential utilityin various applications, such as film production.

SUMMARY OF INVENTION

The present invention is directed toward a film comprising polyalpha-1,3-glucan ester having at least one of: (a) a tear resistance ofat least about 0.1 gf/mil; or (b) a haze of less than about 20%.

In another embodiment, the present invention is directed toward a methodto prepare a poly alpha-1,3-glucan ester film comprising: (a) providingpoly alpha-1,3-glucan ester; (b) contacting the poly alpha-1,3-glucanester of (a) with a solvent to make a solution of poly alpha-1,3-glucanester; (c) applying the solution of poly alpha-1,3-glucan ester on asurface; and (d) allowing the solvent to evaporate to provide the polyalpha-1,3-glucan ester film.

DETAILED DESCRIPTION OF INVENTION

The disclosures of all patent and non-patent literature cited herein areincorporated herein by reference in their entirety.

As used herein, the term “invention” or “disclosed invention” is notmeant to be limiting, but applies generally to any of the inventionsdefined in the claims or described herein. These terms are usedinterchangeably herein.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and“glucan polymer” are used interchangeably herein. Poly alpha-1,3-glucanis a polymer comprising glucose monomeric units linked together byglycosidic linkages, wherein at least about 50% of the glycosidiclinkages are alpha-1,3-glycosidic linkages. Poly alpha-1,3-glucan is atype of polysaccharide. The structure of poly alpha-1,3-glucan can beillustrated as follows:

The poly alpha-1,3-glucan that can be used for preparing polyalpha-1,3-glucan ester compounds herein can be prepared using chemicalmethods. Alternatively, it can be prepared by extracting it from variousorganisms, such as fungi, that produce poly alpha-1,3-glucan.Alternatively still, poly alpha-1,3-glucan can be enzymatically producedfrom sucrose using one or more glucosyltransferase (gtf) enzymes (e.g.,gtfJ), such as described in U.S. Pat. No. 7,000,000, and U.S. PatentAppl. Publ. Nos. 2013/0244288 and 2013/0244287 (all of which areincorporated herein by reference), for example.

The terms “glucosyltransferase enzyme”, “gtf enzyme”, “gtf enzymecatalyst”, “gtf”, and “glucansucrase” are used interchangeably herein.The activity of a gtf enzyme herein catalyzes the reaction of sucrosesubstrate to make products poly alpha-1,3-glucan and fructose. Otherproducts (byproducts) of a gtf reaction can include glucose (whereglucose is hydrolyzed from the glucosyl-gtf enzyme intermediatecomplex), various soluble oligosaccharides (DP2-DP7), and leucrose(where glucose of the glucosyl-gtf enzyme intermediate complex is linkedto fructose). Leucrose is a disaccharide composed of glucose andfructose linked by an alpha-1,5 linkage. Wild type forms ofglucosyltransferase enzymes generally contain (in the N-terminal toC-terminal direction) a signal peptide, a variable domain, a catalyticdomain, and a glucan-binding domain. A gtf herein is classified underthe glycoside hydrolase family 70 (GH70) according to the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009).

The percentage of glycosidic linkages between the glucose monomer unitsof poly alpha-1,3-glucan used to prepare poly alpha-1,3-glucan estercompounds herein that are alpha-1,3 is at least about 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer value between50% and 100%). In such embodiments, accordingly, poly alpha-1,3-glucanhas less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%(or any integer value between 0% and 50%) of glycosidic linkages thatare not alpha-1,3.

Poly alpha-1,3-glucan used to produce poly alpha-1,3-glucan estercompounds herein is preferably linear/unbranched. In certainembodiments, poly alpha-1,3-glucan has no branch points or less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as apercent of the glycosidic linkages in the polymer. Examples of branchpoints include alpha-1,6 branch points, such as those present in mutanpolymer.

The terms “glycosidic linkage” and “glycosidic bond” are usedinterchangeably herein and refer to the type of covalent bond that joinsa carbohydrate (sugar) molecule to another group such as anothercarbohydrate. The term “alpha-1,3-glycosidic linkage” as used hereinrefers to the type of covalent bond that joins alpha-D-glucose moleculesto each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.This linkage is illustrated in the poly alpha-1,3-glucan structureprovided above. Herein, “alpha-D-glucose” is referred to as “glucose”.

The terms “poly alpha-1,3-glucan ester compound”, “poly alpha-1,3-glucanester”, and “poly alpha-1,3-glucan ester derivative” are usedinterchangeably herein. A poly alpha-1,3-glucan ester compound hereincan be represented by the structure:

Regarding the formula of this structure, n can be at least 6, and each Rcan independently be a hydrogen atom (H) or an acyl group. A polyalpha-1,3-glucan ester compound herein has a degree of substitution ofabout 0.05 to about 3.0.

Poly alpha-1,3-glucan ester compounds disclosed herein are synthetic,man-made compounds.

A poly alpha-1,3-glucan ester compound is termed an “ester” herein byvirtue of comprising the substructure —C_(G)—O—CO—C—, where “—C_(G)—”represents carbon 2, 4, or 6 of a glucose monomeric unit of a polyalpha-1,3-glucan ester compound, and where “—CO—C—” is comprised in theacyl group.

An “acyl group” group herein can be an acetyl group (—CO—CH₃), propionylgroup (—CO—CH₂—CH₃), butyryl group (—CO—CH₂—CH₂—CH₃), pentanoyl group(—CO—CH₂—CH₂—CH₂—CH₃), hexanoyl group (—CO—CH₂—CH₂—CH₂—CH₂—CH₃),heptanoyl group (—CO—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃), or octanoyl group(—CO—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃), for example. The carbonyl group(—CO—) of the acyl group is ester-linked to carbon 2, 4, or 6 of aglucose monomeric unit of a poly alpha-1,3-glucan ester compound.

Regarding nomenclature, a poly alpha-1,3-glucan ester compound can bereferenced herein by referring to the organic acid(s) corresponding withthe acyl group(s) in the compound. For example, an ester compoundcomprising acetyl groups can be referred to as a poly alpha-1,3-glucanacetate, an ester compound comprising propionyl groups can be referredto as a poly alpha-1,3-glucan propionate, and an ester compoundcomprising butyryl groups can be referred to as a poly alpha-1,3-glucanbutyrate. However, this nomenclature is not meant to refer to the polyalpha-1,3-glucan ester compounds herein as acids per se.

“Poly alpha-1,3-glucan triacetate” herein refers to a polyalpha-1,3-glucan ester compound with a degree of substitution by acetylgroups of 2.75 or higher.

The terms “poly alpha-1,3-glucan monoester” and “monoester” are usedinterchangeably herein. A poly alpha-1,3-glucan monoester contains onlyone type of acyl group. Examples of such monoesters are polyalpha-1,3-glucan acetate (comprises acetyl groups) and polyalpha-1,3-glucan propionate (comprises propionyl groups).

The terms “poly alpha-1,3-glucan mixed ester” and “mixed ester” are usedinterchangeably herein. A poly alpha-1,3-glucan mixed ester contains twoor more types of an acyl group. Examples of such mixed esters are polyalpha-1,3-glucan acetate propionate (comprises acetyl and propionylgroups) and poly alpha-1,3-glucan acetate butyrate (comprises acetyl andbutyryl groups).

The terms “reaction”, “reaction composition”, and “esterificationreaction” are used interchangeably herein and refer to a reactioncomprising poly alpha-1,3-glucan, at least one acid catalyst, at leastone acid anhydride and at least one organic acid. The reaction issubstantially anhydrous. A reaction is placed under suitable conditions(e.g., time, temperature) for esterification of one or more hydroxylgroups of the glucose units of poly alpha-1,3-glucan with an acyl groupfrom at least the acid anhydride, thereby yielding a polyalpha-1,3-glucan ester compound.

The terms “substantially anhydrous” and “anhydrous” are usedinterchangeably herein. Substantially anhydrous conditions areconditions in which there is less than about 1.5 wt % or 2.0 wt % water.Such conditions may characterize a reaction or a reaction component, forexample.

Herein, a poly alpha-1,3-glucan that is “acid-exchanged” has beentreated with acid to remove water from the poly alpha-1,3-glucan. Anacid-exchange process for producing acid-exchanged poly alpha-1,3-glucancan comprise one or more treatments in which the glucan is placed in anacid (e.g., organic acid) and then removed from the acid.

The term “acid catalyst” as used herein refers to any acid thataccelerates progress of an esterification reaction. Examples of acidcatalysts are inorganic acids such as sulfuric acid (H₂SO₄) andperchloric acid (HClO₄).

The term “acid anhydride” as used herein refers to an organic compoundthat has two acyl groups bound to the same oxygen atom. Typically, anacid anhydride herein has the formula (R—CO)₂O, where R is a saturatedlinear carbon chain (up to seven carbon atoms). Examples of acidanhydrides are acetic anhydride [(CH₃—CO)₂O], propionic anhydride[(CH₃—CH₂—CO)₂O] and butyric anhydride [(CH₃—CH₂—CH₂—CO)₂O].

The terms “organic acid” and “carboxylic acid” are used interchangeablyherein. An organic acid has the formula R—COOH, where R is an organicgroup and COOH is a carboxylic group. The R group herein is typically asaturated linear carbon chain (up to seven carbon atoms). Examples oforganic acids are acetic acid (CH₃—COOH), propionic acid (CH₃—CH₂—COOH)and butyric acid (CH₃—CH₂—CH₂—COOH).

The term “degree of substitution” (DoS) as used herein refers to theaverage number of hydroxyl groups substituted in each monomeric unit(glucose) of a poly alpha-1,3-glucan ester compound. Since there arethree hydroxyl groups in each monomeric unit in poly alpha-1,3-glucan,the DoS in a poly alpha-1,3-glucan ester compound herein can be nohigher than 3.

“Contacting” herein can be performed by any means known in the art, suchas dissolving, mixing, shaking, or homogenization, for example. Wherethree or more reaction components are contacted with each other, suchcontacting can be done all at once or in stages (e.g., two componentsmixed before mixing in a third component).

The “molecular weight” of poly alpha-1,3-glucan and polyalpha-1,3-glucan ester compounds herein can be represented asnumber-average molecular weight (M_(n)) or as weight-average molecularweight (M_(w)). Alternatively, molecular weight can be represented asDaltons, grams/mole, DPw (weight average degree of polymerization), orDPn (number average degree of polymerization). Various means are knownin the art for calculating these molecular weight measurements, such ashigh-pressure liquid chromatography (HPLC), size exclusionchromatography (SEC), or gel permeation chromatography (GPC).

The terms “percent by volume”, “volume percent”, “vol %” and “v/v %” areused interchangeably herein. The percent by volume of a solute in asolution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)” and“weight-weight percentage (% w/w)” are used interchangeably herein.Percent by weight refers to the percentage of a material on a mass basisas it is comprised in a composition, mixture or solution.

The terms “increased”, “enhanced” and “improved” are usedinterchangeably herein. These terms may refer to, for example, aquantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%,or 200% (or any integer between 1% and 200%) more than the quantity oractivity for which the increased quantity or activity is being compared.

Embodiments of the disclosed invention concern a composition comprisinga poly alpha-1,3-glucan ester compound represented by the structure:

Regarding the formula of this structure, n can be at least 6, and each Rcan independently be an H or an acyl group. Furthermore, the polyalpha-1,3-glucan ester compound has a degree of substitution of about0.05 to about 3.0.

Each R group in the formula of the poly alpha-1,3-glucan ester compoundcan independently be an H or an acyl group. An acyl group herein can bean acetyl group, propionyl group, butyryl group, pentanoyl group,hexanoyl group, heptanoyl group, or octanoyl group, for example. Thus,an acyl group can comprise a chain of 2 to 8 carbons; this chainpreferably has no branches.

Poly alpha-1,3-glucan ester compounds in certain embodiments disclosedherein may contain one type of acyl group. For example, one or more Rgroups ester-linked to the glucose group in the above formula may be apropionyl group; the R groups in this particular example would thusindependently be hydrogen and propionyl groups. As another example, oneor more R groups ester-linked to the glucose group in the above formulamay be an acetyl group; the R groups in this particular example wouldthus independently be hydrogen and acetyl groups. Certain embodiments ofpoly alpha-1,3-glucan ester compounds herein do not have a DoS by acetylgroups of 2.75 or more.

Alternatively, poly alpha-1,3-glucan ester compounds disclosed hereincan contain two or more different types of acyl groups. Examples of suchcompounds contain two different acyl groups, such as (i) acetyl andpropionyl groups (poly alpha-1,3-glucan acetate propionate, where Rgroups are independently H, acetyl, or propionyl), or (ii) acetyl andbutyryl groups (poly alpha-1,3-glucan acetate butyrate, where R groupsare independently H, acetyl, or butyryl).

The poly alpha-1,3-glucan ester compound has a degree of substitution(DoS) of about 0.05 to about 3.0. Alternatively, the DoS of a polyalpha-1,3-glucan ester compound disclosed herein can be about 0.2 toabout 2.0. Alternatively still, the DoS can be at least about 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. Itwould be understood by those skilled in the art that since a polyalpha-1,3-glucan ester compound disclosed herein has a degree ofsubstitution between about 0.05 to about 3.0, the R groups of thecompound cannot only be hydrogen.

The wt % of one or more acyl groups in a poly alpha-1,3-glucan estercompound herein can be referred to instead of referencing a DoS value.For example, the wt % of an acyl group in a poly alpha-1,3-glucan estercompound can be at least about 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%.

The percentage of glycosidic linkages between the glucose monomer unitsof the poly alpha-1,3-glucan ester compound that are alpha-1,3 is atleast about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%(or any integer between 50% and 100%). In such embodiments, accordingly,the compound has less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%,2%, 1%, or 0% (or any integer value between 0% and 50%) of glycosidiclinkages that are not alpha-1,3.

The backbone of a poly alpha-1,3-glucan ester compound disclosed hereinis preferably linear/unbranched. In certain embodiments, the compoundhas no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% branch points as a percent of the glycosidic linkages in thepolymer. Examples of branch points include alpha-1,6 branch points.

The formula of a poly alpha-1,3-glucan ester compound in certainembodiments can have an n value of at least 6. Alternatively, n can havea value of at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300,3400, 3500, 3600, 3700, 3800, 3900, or 4000 (or any integer between 10and 4000).

The molecular weight of a poly alpha-1,3-glucan ester compound disclosedherein can be measured as number-average molecular weight (M_(n)) or asweight-average molecular weight (M_(w)). Alternatively, molecular weightcan be measured in Daltons or grams/mole. It may also be useful to referto the DP_(w) (weight average degree of polymerization) or DP_(n)(number average degree of polymerization) of the poly alpha-1,3-glucanpolymer component of the compound.

The M_(n) or M_(w) of poly alpha-1,3-glucan ester compounds disclosedherein may be at least about 1000. Alternatively, the M_(n) or M_(w) canbe at least about 1000 to about 600000. Alternatively still, the M_(n)or M_(w) can be at least about 10000, 25000, 50000, 75000, 100000,125000, 150000, 175000, 200000, 225000, 250000, 275000, or 300000 (orany integer between 10000 and 300000), for example.

A poly alpha-1,3-glucan ester in certain embodiments can have a DoS byacetyl groups up to about 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30,2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90,2.95, or 3.00. Thus, for example, the DoS by acetyl groups can be up toabout 2.00-2.40, 2.00-2.50, or 2.00-2.65. As other examples, the DoS byacetyl groups can be about 0.05 to about 2.60, about 0.05 to about 2.70,about 1.2 to about 2.60, or about 1.2 to about 2.70. Such polyalpha-1,3-glucan esters can be a monoester or a mixed ester.

A poly alpha-1,3-glucan ester in certain embodiments can have a wt % ofpropionyl groups up to about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, or 55%. Such poly alpha-1,3-glucan esters can be amonoester or a mixed ester. Regarding mixed esters, polyalpha-1,3-glucan acetate propionate can have a wt % of acetyl groups upto about 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, and a wt % ofpropionyl groups as per any of the propionyl wt %'s listed above, forexample.

A poly alpha-1,3-glucan ester in certain embodiments can have a wt % ofbutyryl groups up to about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, or 60%. A poly alpha-1,3-glucan ester in other embodiments can havea DoS by butyryl groups up to about 0.80, 0.85, 0.90, 0.95, 1.00, 1.05,1.10, 1.15, or 1.20. Such poly alpha-1,3-glucan esters can be amonoester or a mixed ester. Regarding mixed esters, polyalpha-1,3-glucan acetate butyrate can have a wt % of acetyl groups up toabout 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, or 36%, and a wt % of butyryl groupsas per any of the butyryl wt %'s listed above, for example.

The disclosed invention also concerns a method for producing a polyalpha-1,3-glucan ester compound. This method comprises: contacting polyalpha-1,3-glucan in a reaction that is substantially anhydrous with atleast one acid catalyst, at least one acid anhydride, and at least oneorganic acid, wherein an acyl group derived from the acid anhydride isesterified to the poly alpha-1,3-glucan thereby producing a polyalpha-1,3-glucan ester compound represented by the structure:

wherein(i) n is at least 6,(ii) each R is independently an H or the acyl group, and(iii) the compound has a degree of substitution of about 0.05 to about3.0. A poly alpha-1,3-glucan ester produced by this method canoptionally be isolated.

A poly alpha-1,3-glucan is contacted with at least one acid catalyst, atleast one acid anhydride, and at least one organic acid in a reactionthat is substantially anhydrous. A substantially anhydrous reactionherein contains no water or less than about 0.05, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2.0 wt % water. Substantially anhydrous conditions can beobtained by using reaction components that are substantially anhydrous.Reaction components that are not substantially anhydrous may be used forpreparing a reaction, but only in amounts such that the final reactionpreparation is substantially anhydrous.

Enzymatically produced preparations of poly alpha-1,3-glucan that can beused in the disclosed esterification reaction typically contain water.This poly alpha-1,3-glucan can be acid-exchanged to remove water therebyrendering the glucan to be substantially anhydrous. In certainembodiments, poly alpha-1,3-glucan can be acid-exchanged with an organicacid (e.g., acetic, propionic, or butyric acid) before contacting step(a) to remove water from the poly alpha-1,3-glucan. An acid-exchangeprocess herein can comprise boiling poly alpha-1,3-glucan in water,removing most of the water by any physical means (e.g., filtration,decantation, and/or drying), placing the glucan in an organic acid, andthen removing the organic acid by filtration and/or decantation.Treatment with an organic acid can comprise stirring the glucan in theacid, and can be performed one, two, or more times. The amount oforganic acid used in each treatment can be at least about 2 to 20 times,or 2 to 10 times, the amount of poly alpha-1,3-glucan being treated, forexample.

Poly alpha-1,3-glucan is contacted with at least one acid catalyst inthe disclosed reaction. An acid catalyst can be an inorganic acid incertain embodiments. Examples of an inorganic acid catalyst that can beincluded in a reaction herein are sulfuric acid and perchloric acid.Other examples of inorganic acid catalysts include hydrochloric,phosphoric, nitric, boric, hydrofluoric, hydrobromic, sulfonic, anymineral acid, and any combination thereof. An acid catalyst herein cantypically be obtained commercially in a concentrated (e.g., >95%, 96%,97%, 98%, or 99% pure) and/or substantially anhydrous form. For example,sulfuric acid for use in a reaction herein can be at least about 95-98%pure. Alternatively, an acid catalyst can be provided in solution withan organic acid such as acetic acid. An example of such a solution isperchloric acid (0.1 N) in acetic acid. The amount of acid catalyst in areaction can be at least about 0.005, 0.0075, 0.01, 0.025, 0.05, 0.075,0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, or 2.0 wt %, for example.

Poly alpha-1,3-glucan is contacted with at least one acid anhydride inthe disclosed reaction. Examples of an acid anhydride that can beincluded in a reaction herein include acetic anhydride, propionicanhydride, butyric anhydride, pentanoic anhydride, hexanoic anhydride,heptanoic anhydride, octanoic anhydride and phthalic anhydride. Anycombination of these can be used in a reaction herein (e.g., acetic andpropionic anhydrides, acetic and butyric anhydrides, propionic andbutyric anhydrides). An acid anhydride herein can typically be obtainedcommercially in a concentrated (e.g., >95%, 96%, 97%, 98%, or 99% pure)and/or substantially anhydrous form. For example, acetic anhydride,propionic anhydride and/or butyric anhydride for use in a reactionherein can be at least about 97%, 98%, or 99% pure. The amount of acidanhydride in a reaction can be at least about 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, or 70 wt % (or any integer value between 10 and70 wt %), for example. In certain embodiments, the amount of aceticanhydride in a reaction can be at least about 20-45 wt %. The amount ofpropionic or butyric anhydride in other embodiments can be at leastabout 40-50 wt %.

Poly alpha-1,3-glucan is contacted with at least one organic acid in thedisclosed reaction. Examples of an organic acid that can be included ina reaction herein include acetic acid, propionic acid, butyric acid,pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid andphthalic acid. The amount of organic acid in a reaction can be at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80wt % (or any integer value between 5 and 80 wt %), for example.

Typically, one or more acid anhydrides used in a reaction herein areselected based on the type of esterification desired. As examples, ifesterification of poly alpha-1,3-glucan with acetyl groups, propionylgroups and/or butyryl groups is desired, then acetic anhydride,propionic anhydride and/or butyric anhydride, respectively, is/areincluded in the reaction accordingly. The selected acid anhydride(s) isthe main source of acyl groups in the disclosed esterification process.That being said, acyl groups for esterification can also be derived fromone or more organic acids included in the reaction. For example, anacetyl group, propionyl group, butyryl group, pentanoyl group, hexanoylgroup, heptanoyl group, and octanoyl group can be derived from,respectively, acetic acid, propionic acid, butyric acid, pentanoic acid,hexanoic acid, heptanoic acid, and octanoic acid. Reactions containing aparticular acid anhydride typically also contain the organic acidcorresponding to the acid anhydride.

In certain embodiments of the disclosed reaction, the acid anhydride isone or more of acetic anhydride, propionic anhydride, or butyricanhydride; and the organic acid is one or more of acetic acid, propionicacid, or butyric acid. Combinations of (i) acetic anhydride and aceticacid can be used to prepare poly alpha-1,3-glucan acetate; (ii)propionic anhydride and propionic acid can be used to prepare polyalpha-1,3-glucan propionate; (iii) butyric anhydride and butyric acidcan be used to prepare poly alpha-1,3-glucan butyrate; (iv) propionicanhydride, propionic acid, acetic anhydride and optionally acetic acidcan be used to prepare poly alpha-1,3-glucan acetate propionate; (v)propionic anhydride, propionic acid and acetic acid can be used toprepare poly alpha-1,3-glucan acetate propionate; (vi) butyricanhydride, butyric acid, acetic anhydride and optionally acetic acid canbe used to prepare poly alpha-1,3-glucan acetate butyrate; and (vii)butyric anhydride, butyric acid and acetic acid can be used to preparepoly alpha-1,3-glucan acetate butyrate, for example. In reactionscontaining acetic acid along with propionic acid or butyric acid, theamount of acetic acid can be about 5-10, 5-20, or 5-30 wt %, forexample.

Reactions for producing mixed esters (e.g., poly alpha-1,3-glucanacetate propionate, poly alpha-1,3-glucan acetate butyrate) typicallycontain more of an acid anhydride having an acyl group for which ahigher DoS is desired, and less of an acid anhydride and/orcorresponding organic acid for which a lower DoS is desired. Forexample, to produce a poly alpha-1,3-glucan acetate propionate with ahigher DoS of propionyl groups compared to acetyl groups, more propionicanhydride is included in a reaction compared to the amount of aceticanhydride and/or acetic acid. DoS in mixed esters may also be modulatedby the order in which acid anhydrides are added to a reaction alreadycontaining an acid catalyst. For example, one may expect a higher DoSwith propionyl groups if propionic anhydride is added before aceticanhydride (to a preparation already containing acid catalyst) whenpreparing a reaction to produce poly alpha-1,3-glucan acetatepropionate.

An acid anhydride selected for a reaction herein can correspond with theorganic acid used to prepare acid-exchanged poly alpha-1,3-glucan. Forexample, if a reaction will include propionic anhydride, then an acidexchange process can be performed with propionic acid. Alternatively, anacid anhydride selected for a reaction herein can differ from theorganic acid used to prepare acid-exchanged poly alpha-1,3-glucan. Forexample, if a reaction will include propionic anhydride, then an acidexchange process can be performed with acetic acid.

A reaction herein can comprise components in addition to polyalpha-1,3-glucan, acid catalyst, acid anhydride, and organic acid. Forexample, one or more organic solvents can be included in a reaction,such as methylene chloride. An organic solvent such a methylene chloridecan be included at about 30-40 wt % in a reaction (e.g., producingglucan triacetate), for example.

The components of a reaction herein can be added together in any order.For example, poly alpha-1,3-glucan, acid catalyst and organic acid canfirst be mixed together, afterwhich acid anhydride can be added to themixture. As another example, acid anhydride and organic acid can firstbe mixed together, afterwhich poly alpha-1,3-glucan and acid catalystcan be added to the mixture. As yet another example, acid catalyst andorganic acid can first be mixed together, afterwhich polyalpha-1,3-glucan and acid anhydride can be added to the mixture. Incertain embodiments, poly alpha-1,3-glucan and another component (e.g.,acid catalyst or acid anhydride) are added in sequential order to amixture containing the other reaction components.

Cooling can be applied during various stages of preparing a reactionherein. The terms “cool” and “chill” are used interchangeably herein andrefer to decreasing the temperature of a reaction or mixture to a lowertemperature. Cooling can be performed by any means known in the art,such as with an ice bath or industrial cooling system. Step (a) ofpreparing a reaction can comprise cooling the reaction after itspreparation (i.e., containing all of poly alpha-1,3-glucan, acidcatalyst, acid anhydride and organic acid), such as to about 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20° C., or about 12-18° C. Alternatively,step (a) can comprise cooling (e.g., to any of the precedingtemperatures) a mixture containing poly alpha-1,3-glucan, acid catalystand organic acid, and then adding acid anhydride to the cooled mixture.Alternatively still, step (a) can comprise cooling (e.g., to any of thepreceding temperatures) a mixture containing acid anhydride and organicacid, and then adding poly alpha-1,3-glucan and acid catalyst to thecooled mixture. Alternatively still, step (a) can comprise cooling(e.g., to any of the preceding temperatures) a mixture containing acidcatalyst and organic acid, and then adding poly alpha-1,3-glucan andacid anhydride to the cooled mixture. A reaction can optionally be heldat any of the preceding cooler temperature points for about 1-10 minutesafter its initial preparation.

A reaction can then be (i) placed under ambient temperature conditionswithout direct application of heat, and/or (ii) directly heated usingany means known in the art (e.g., water bath, industrial or electricheater). Ambient temperature conditions can be held for up to about 30,60, 120, 240, 360, or 480 minutes (or any integer value between 30 and480 minutes), for example. Alternatively, ambient temperature conditionscan be held for up to about 24, 48, or 72 hours. The term “ambienttemperature” as used herein refers to a temperature between about 15-30°C. or 20-25° C. (or any integer between 15 and 30° C.). Reaction heatingcan be up to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80° C. (orany integer value between 30 and 80° C.), about 30-60° C., or about30-50° C., for example. Such heating can be done is stages, if desired.For example, a reaction can first be heated to about 35° C., and thenheated to about 39-50° C. A maximum reaction temperature (e.g., about36-43° C.) may be applied to avoid excess degradation of polyalpha-1,3-glucan ester molecular weight in certain embodiments, such aswhen producing poly alpha-1,3-glucan propionate, poly alpha-1,3-glucanacetate propionate, or poly alpha-1,3-glucan acetate butyrate. Thetemperature after heating to any of the preceding temperatures can bemaintained for about 20-30, 20-40, 20-60 minutes, or up to about 40, 60,80, 100, 120, or 140 minutes, for example. When heating is done instages, the first temperature point(s) can be held for about 20-40minutes, for example. In embodiments in which a reaction is placed underambient temperature conditions without direct application of heat, thereaction can subsequently be heated, if desired, to any of the precedingtemperatures and time periods. A reaction typically does not contain anysolid material, but may be viscous, after any of the above temperaturetreatments (ambient temperature and/or heating).

A reaction can optionally be cooled after any of the above temperaturetreatments (ambient temperature and/or heating). For example, a reactioncan be cooled to about 18, 19, 20, 21, 22, 23, 24, or 25° C., about20-30° C., or about 20-40° C. A reaction that was heated to 60-80° C.can typically be cooled to about 35-45° C. The temperature of a reactionupon cooling can be held for about 5-10 minutes, for example.

Optionally, a reaction herein can be maintained under an inert gas(e.g., nitrogen). As used herein, the term “inert gas” refers to a gaswhich does not undergo chemical reactions under a set of givenconditions, such as those disclosed for preparing a reaction herein.

A reaction can optionally be quenched after any of the above temperaturetreatments (ambient temperature and/or heating) and cooling treatments.Quenching of a reaction can be accomplished by adding acid, base, orcertain salts to the reaction. Various acids, bases and salts useful forquenching a reaction include, but are not limited to, acetic acid (e.g.,˜50-70 wt %), any other mineral or organic acid (e.g., ˜50-70 wt %),magnesium acetate (e.g., ˜20-25 wt %), sodium hydroxide, potassiumhydroxide, calcium hydroxide, sodium bicarbonate, sodium carbonate andcombinations thereof. In certain embodiments of producing polyalpha-1,3-glucan acetate, a reaction is quenched with acetic acid (e.g.,˜50 or 70 wt %) or magnesium acetate (e.g., ˜20-25 wt %).

A quenched reaction can optionally be heated to about 40° C. to 150° C.for up to 48 hours. For example, a quenched reaction can be heated toabout 100° C. for up to about 20-40 minutes (e.g., 25-30 minutes), suchas in a process for producing poly alpha-1,3-glucan acetate. Optionally,water may be added to a reaction (quenched or not quenched), which isthen heated to about 40° C. to 150° C. (e.g., ˜100° C.) for up to about20-40 minutes (e.g., 25-30 minutes) to reduce DoS of acyl groups byhydrolysis. Such a heating/water-treatment step may be useful forreducing DoS in a process for producing poly alpha-1,3-glucan acetate.

A poly alpha-1,3-glucan ester compound produced by a reaction herein canbe precipitated using an agent that is a non-solvent for the polyalpha-1,3-glucan ester compound. For example, deionized water and/ormethanol can be added to a reaction solution in an amount sufficient toprecipitate a poly alpha-1,3-glucan ester compound. Precipitation hereincan further comprise mixing the reaction solution and non-solvent by anymeans known in the art, such as with an air-powered blender.

Precipitated poly alpha-1,3-glucan ester compound can optionally beneutralized by washing it with water two or more times, followed by awash in a bicarbonate (e.g., sodium bicarbonate) solution (e.g., ˜5 wt%). The ester compound can then be washed one, two or more times withwater until neutral pH is achieved. Alternatively, precipitated polyalpha-1,3-glucan ester compound can be washed with water and base (e.g.,diluted alkaline hydroxide such as sodium hydroxide, calcium hydroxide,or potassium hydroxide) to achieve a neutral pH, optionally followed bywashing with water. The term “neutral pH” as used herein refers to a pHthat is neither substantially acidic or basic (e.g., a pH of about 6-8,or about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0).

A poly alpha-1,3-glucan ester produced in the disclosed reaction can beisolated. The above precipitation process can be a step in an isolationprocess. Isolation can be performed with precipitated product before orafter neutralization and/or washing steps using a funnel, centrifuge,press filter, or any other method or equipment known in the art thatallows removal of liquids from solids. An isolated poly alpha-1,3-glucanester product can be dried using any method known in the art, such asvacuum drying, air drying (e.g., −16-35° C.), or freeze drying.

Any of the above esterification reactions can be repeated using a polyalpha-1,3-glucan ester product as the starting material for furthermodification. This approach may be suitable for increasing the DoS of anacyl group, and/or adding one or more different acyl groups to the esterproduct.

The structure, molecular weight and DoS of a poly alpha-1,3-glucan esterproduct can be confirmed using various physiochemical analyses known inthe art such as NMR spectroscopy and size exclusion chromatography(SEC).

The percentage of glycosidic linkages between the glucose monomer unitsof poly alpha-1,3-glucan used to prepare poly alpha-1,3-glucan estercompounds herein that are alpha-1,3 is at least about 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer value between50% and 100%). In such embodiments, accordingly, poly alpha-1,3-glucanhas less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%(or any integer value between 0% and 50%) of glycosidic linkages thatare not alpha-1,3.

Poly alpha-1,3-glucan used to prepare poly alpha-1,3-glucan estercompounds herein is preferably linear/unbranched. In certainembodiments, poly alpha-1,3-glucan has no branch points or less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as apercent of the glycosidic linkages in the polymer. Examples of branchpoints include alpha-1,6 branch points.

The M_(n) or M_(w) of poly alpha-1,3-glucan used to prepare polyalpha-1,3-glucan ester compounds herein may be at least about 500 toabout 300000. Alternatively still, M_(n) or M_(w) can be at least about10000, 25000, 50000, 75000, 100000, 125000, 150000, 175000, 200000,225000, 250000, 275000, or 300000 (or any integer between 10000 and300000), for example.

A process is disclosed herein for producing poly alpha-1,3-glucanacetate with a DoS of 0.05 to 2.70 using poly alpha-1,3-glucantriacetate. The triacetate used in this process can be producedaccording to any of the above processes, for example. This processcomprises: contacting poly alpha-1,3-glucan triacetate with acetic acidand water to form a preparation, and applying steam pressure of about3-10 kg/cm² to the preparation to raise its temperature up to about 260°C. This process results in a poly alpha-1,3-glucan acetate having a DoSof 0.05 to 2.70. Such reduction in DoS results from hydrolysis of aportion of the acetyl groups of the poly alpha-1,3-glucan triacetate. Apoly alpha-1,3-glucan acetate produced by this method can optionally beisolated.

Poly alpha-1,3-glucan triacetate can optionally be washed and/or have aneutral pH prior to use in this process. Poly alpha-1,3-glucantriacetate can be contacted with acetic acid and water by firstdissolving the glucan triacetate in acetic acid, and then adding waterto this solution. In certain embodiments, the amount of acetic acid inthe preparation can be about 75, 80, 85, or 90 wt %, and the amount ofwater in the preparation can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 wt %.

A preparation containing poly alpha-1,3-glucan triacetate, acetic acidand water is then subjected to steam pressure of about 3-10 kg/cm² toraise its temperature up to about 260° C. This step can be optionally becarried out in a pressure vessel such as a Parr reactor, autoclave, orany other pressure vessel known in the art. A steam pressure of about 4,5, or 6 kg/cm², for example, can be used to raise the temperature of thepreparation to about 140-160° C. (e.g., 150° C.). This elevatedtemperature can be held for about 30, 40, 50, 60, or 70 minutes,afterwhich the applied pressure can be increased further to about 7, 8,or 9 kg/cm². After reaching this elevated pressure, the temperature canbe cooled to ambient temperature.

Poly alpha-1,3-glucan acetate having a DoS of 0.05 to 2.70 can beisolated from the pressure-/heat-treated preparation using any of theprecipitation, washing and isolation steps disclosed above.

Poly alpha-1,3-glucan esters formed using various methods describedabove can be used to prepare various types of films. The polyalpha-1,3-glucan esters prepared according to the disclosed methods canbe dissolved in one or more solvents to provide a solution of polyalpha-1,3-glucan ester. As used herein, the term “solution of polyalpha-1,3-glucan ester” refers to poly alpha-1,3-glucan ester dissolvedin one or more solvents. The solvents useful for this purpose include,but are not limited to, methylene chloride (dichloromethane), methanol,chloroform, tetrachloroethane, formic acid, acetic acid, nitrobenzene,bromoform, pyridine, dioxane, ethanol, acetone, alcohols, aromaticcompounds such as monochlorobenzene, benzene and toluene, esters such asethyl acetate and propyl acetate, ethers such as tetrahydrofuran, methylcellosolve and ethylene glycol monomethyl ether or combinations thereof.In an embodiment poly alpha-1,3-glucan acetate is dissolved in acetoneto prepare a solution of poly alpha-1,3-glucan acetate. This solutioncan then be applied to a surface and the solvent is allowed to evaporateto form a film of desired thickness. The surfaces suitable for thisapplication can be, but are not limited to, glass, Teflon®, plastic, orvarious types of substrates. Methods to make films from theabove-mentioned solution, which are well known in the art, include butnot limited to solution casting, spin coating, thermal and regularspraying. In an embodiment, the solution of poly alpha-1,3-glucan esteris cast on a glass plate.

The tear resistance, tensile strength and temperature stability of thepoly alpha-1,3-glucan ester films can be determined by methods wellknown in the art. As used herein, the term “tear resistance” is definedas a measure of how well a film can withstand the effects of tearing.The term “tensile strength”, as used herein, refers to the maximumtension a material can withstand without tearing. The suitable tearresistance for a poly alpha-1,3-glucan ester film disclosed herein canbe at least 0.1 gf/mil. The tensile strength of the film suitable forthe disclosed invention can be at least 4 kgf/mm². In an embodiment, thetear resistance of the poly alpha-1,3-glucan diacetate film is 2-2.4gf/mil and the tensile strength is 3.97-4.8 kgf/mm². In anotherembodiment, the tear resistance is 1.4-3.1 gf/mil and the tensilestrength is 4.98-6.44 kgf/mm².

The haze and transmittance of the poly alpha-1,3-glucan ester film canbe determined by methods well known in the art. As used herein, the term“haze” refers to the percentage of light that is deflected more than 2.5degrees from the incoming light direction. Low haze values correspond tobetter clarity. The term “transmittance” as used herein, refers to thefraction of incident light at a specified wavelength that passes througha film. The suitable film for the poly alpha-1,3-glucan acetate film inthis application can have a haze up to 20% and the transmittance of atleast 80%. In an embodiment, the haze is 6.2% and the transmittance is94.6%.

The speed with which the film is produced can be increased by adding aweak solvent, such as methanol and cyclohexane, ethanol and n-butanol orabundant methanol or ethanol in addition to methylene chloride, into thepoly alpha-1,3-glucan ester solution to accelerate the solidificationspeed. One can restrain planar orientation degree and crystallizationdegree by controlling the surface temperature and shrinkage percentageof a film.

EXAMPLES

The disclosed invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating certainpreferred aspects of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usesand conditions.

ABBREVIATIONS

“mL” is milliliter(s); “g” is gram(s); “DI water” is deionized water;“μL” is microliter(s); “° C.” is degrees Celsius; “mg” is milligram(s);“TFA” is trifluoroacetic acid; “Hz” is Hertz; “MHz” is mega Hertz; “ppm”is parts per million; “HFIP” is hexafluoro-2-propanol; “TFA-d” isdeuterated trifluoroacetic acid, “kgf” is kilogram force.

Materials

Sulfuric acid, acetic acid and sodium bicarbonate were from EMDChemicals (Billerica, Mass.). Acetic anhydride was from Acros Organics(Pittsburgh, Pa.). Butyric acid, butyric anhydride, propionic anhydrideand 0.1 N perchloric acid in acetic acid were from Sigma Aldrich (St.Louis, Mo.). Propionic acid was from JT Baker (Center Valley, Pa.).Magnesium acetate was from Alfa Aesar (Ward Hill, Mass.). Unlessotherwise specified, all acids and anhydrides used herein werewater-free or substantially water-free.

Preparation of Poly Alpha-1,3-Glucan

Poly alpha-1,3-glucan was prepared using a gtfJ enzyme preparation asdescribed in U.S. Patent Appl. Publ. No. 2013/0244288, which isincorporated herein by reference in its entirety.

¹H Nuclear Magnetic Resonance (NMR) Method for Determining Degree ofSubstitution of Poly Alpha-1,3-Glucan Acetate Derivatives

Degree of substitution (DoS) in poly alpha-1,3-glucan acetate esterderivatives was determined using ¹H NMR. Approximately 20 mg ofderivative sample was weighed into a vial on an analytical balance. Thevial was removed from the balance and 0.7 mL of TFA-d was added to thevial. A magnetic stir bar was added to the vial and the mixture wasstirred until the solid sample dissolved. Deuterated benzene (C₆D₆), 0.3mL, was then added to the vial to provide a better NMR lock signal thanthe TFA-d would provide. A portion, 0.8 mL, of the solution wastransferred using a glass pipet into a 5-mm NMR tube. A quantitative ¹HNMR spectrum was acquired using an Agilent VNMRS 400 MHz NMRspectrometer equipped with a 5-mm Autoswitchable Quad probe. Thespectrum was acquired at a spectral frequency of 399.945 MHz using aspectral window of 6410.3 Hz, an acquisition time of 1.278 seconds, andan inter-pulse delay of 10 seconds and 124 pulses. The time domain datawere transformed using exponential multiplication of 0.78 Hz.

Two regions of the resulting spectrum were integrated: from 3.1 ppm to6.0 ppm, giving the integral for the seven poly alpha-1,3-glucanprotons, and from 1.4 ppm to 2.7 ppm, giving the integral for the threeacetyl protons. The degree of acetylation was calculated by dividing onethird of the acetyl protons integral area by one seventh of the polyalpha-1,3-glucan protons integral area.

¹H NMR Method for Determining Degree of Substitution of PolyAlpha-1,3-Glucan Propionate Derivatives

DoS in poly alpha-1,3-glucan propionate ester derivatives was determinedusing ¹H NMR. Approximately 20 mg of derivative sample was weighed intoa vial on an analytical balance. The vial was removed from the balanceand 0.7 mL of TFA-d was added to the vial. A magnetic stir bar was addedto the vial and the mixture was stirred until the solid sampledissolved. Deuterated benzene (C₆D₆), 0.3 mL, was then added to the vialto provide a better NMR lock signal than the TFA-d would provide. Aportion, 0.8 mL, of the solution was transferred using a glass pipetinto a 5-mm NMR tube. A quantitative ¹H NMR spectrum was acquired usingan Agilent VNMRS 400 MHz NMR spectrometer equipped with a 5-mmAutoswitchable Quad probe. The spectrum was acquired at a spectralfrequency is 399.945 MHz using a spectral window of 6410.3 Hz, anacquisition time of 1.278 seconds, and an inter-pulse delay of 10seconds and 32 pulses. The time domain data were transformed usingexponential line broadening of 1.0 Hz and the benzene solvent peak wasset to 7.15 ppm.

For poly alpha-1,3-glucan propionate samples, three regions of theresulting spectrum were integrated: from 3.3 ppm to 6.0 ppm, giving theintegral for the seven poly alpha-1,3-glucan protons; from 1.9 ppm to2.7 ppm, giving the integral for the methylene group of the propionylgroup plus the methyl group of the acetyl group; and from 0.8 ppm to 1.3ppm, giving the integral for the methyl group of the propionyl group.

The DoS by propionyl groups was calculated by dividing the integralvalue for the methyl group of the propionyl group by three. The integralvalue of the propionyl group's methylene group was then calculated bymultiplying the integral value for the methyl group of the propionylgroup by 0.666. This value was then subtracted from the integral for theregion of the methylene group of the propionyl group plus the methylgroup of the acetyl group to give the integral value for the acetylgroup's methyl group.

¹H NMR Method for Determining Degree of Substitution of PolyAlpha-1,3-Glucan Mixed Ester Derivatives

DoS in poly alpha-1,3-glucan mixed ester derivatives was determinedusing ¹H NMR. Approximately 20 mg of derivative sample was weighed intoa vial on an analytical balance. The vial was removed from the balanceand 0.7 mL of TFA-d was added to the vial. A magnetic stir bar was addedto the vial and the mixture was stirred until the solid sampledissolved. Deuterated benzene (C₆D₆), 0.3 mL, was then added to the vialto provide a better NMR lock signal than the TFA-d would provide. Aportion, 0.8 mL, of the solution was transferred using a glass pipetinto a 5-mm NMR tube. A quantitative ¹H NMR spectrum was acquired usingan Agilent VNMRS 400 MHz NMR spectrometer equipped with a 5-mmAutoswitchable Quad probe. The spectrum was acquired at a spectralfrequency of 399.945 MHz using a spectral window of 6410.3 Hz, anacquisition time of 1.278 seconds, and inter-pulse delay of 10 secondsand 32 pulses. The time domain data were transformed using exponentialline broadening of 1.0 Hz and the benzene solvent peak was set to 7.15ppm.

For poly alpha-1,3-glucan acetate propionate samples, three regions ofthe resulting spectrum were integrated: from 3.3 ppm to 6.0 ppm, givingthe integral for the seven poly alpha-1,3-glucan protons; from 1.9 ppmto 2.7 ppm giving the integral for the methylene group of the propionylgroup plus the methyl group of the acetyl group; and from 0.8 ppm to 1.3ppm giving the integral for the methyl group of the propionyl group.

The DoS by propionyl groups on the glucan was calculated by dividing theintegral value for the methyl group of the propionyl group by three. Theintegral value of the propionyl group's methylene group was thencalculated by multiplying the integral value for the methyl group of thepropionyl group by 0.666. This value was then subtracted from theintegral for the region of the methylene group of the propionyl groupplus the methyl group of the acetyl group to give the integral value forthe acetyl group's methyl group. Finally, the acetyl group integralvalue was divided by three to obtain the degree of acetylation.

For poly alpha-1,3-glucan acetate butyrate samples, three regions of theresulting spectrum were integrated: from 3.3 ppm to 6.0 ppm giving theintegral for the seven poly alpha-1,3-glucan protons; from 1.9 ppm to2.6 ppm giving the integral for the methylene group alpha to thecarbonyl of the butyryl group plus the methyl group of the acetyl group;and from 0.6 ppm to 1.0 ppm giving the integral for the methyl group ofthe butyryl group.

The DoS by butyryl groups on the glucan was calculated by dividing theintegral value for the methyl group of the butyryl group by three. Theintegral value of the butyryl group's methylene group was thencalculated by multiplying the integral value for the methyl group of thebutyryl group by 0.666. This value was then subtracted from the integralfor the region of the methylene group of the butyryl group plus themethyl group of the acetyl group to give the integral value for theacetyl group's methyl group. Finally, the acetyl group integral valuewas divided by three to obtain the degree of acetylation.

Determination of the Degree of Polymerization

The degree of polymerization (DP) was determined by size exclusionchromatography (SEC). Poly alpha-1,3-glucan ester was dissolved in HFIP(2 mg/mL) with shaking for 4 hours at 45° C. The chromatographic systemused was Alliance™ 2695 separation module from Waters Corporation(Milford, Mass.) coupled with three on-line detectors: a differentialrefractometer 2410 from Waters, a multi-angle light-scatteringphotometer Heleos™ 8+ from Wyatt Technologies (Santa Barbara, Calif.),and a differential capillary viscometer ViscoStar™ from WyattTechnologies. The columns used for SEC were two Shodex (Showa DenkoAmerica, New York) GPC HFIP-806M™ styrene-divinyl benzene columns andone Shodex GPC HFIP-804M™ styrene-divinyl benzene column. The mobilephase was redistilled HFIP with 0.01 M sodium trifluoroacetate.Chromatographic conditions used were 50° C. at column and detectorcompartments, 40° C. at sample and injector compartments, a flow rate of0.5 mL/min, and injection volume of 100 μL. Software packages used fordata reduction were Astra version 6 from Wyatt (triple detection methodwith column calibration).

Example 1 Preparation of Acid-Exchanged Poly Alpha-1,3-Glucan

This Example describes producing acid-exchanged poly alpha-1,3-glucan,which can be used for preparing ester derivatives of polyalpha-1,3-glucan.

Acid-exchanged poly alpha-1,3-glucan was prepared by placing 10 g ofpoly alpha-1,3-glucan in a 250-mL glass beaker with 150 mL of DI water.This mixture was boiled for one hour on a hot plate, afterwhich the polyalpha-1,3-glucan was recovered by vacuum filtration. The polyalpha-1,3-glucan was then subjected to two acid exchange steps ofstirring it with 100 mL of glacial acetic acid at room temperaturefollowed by vacuum filtration, thereby providing acid-exchanged polyalpha-1,3-glucan.

Other forms of acid-exchanged poly alpha-1,3-glucan were also preparedby following the above process, but using propionic acid or butyric acidinstead of acetic acid.

Acid-exchanged poly alpha-1,3-glucan prepared by these techniques wasused in certain of the following examples to prepare various polyalpha-1,3-glucan ester derivatives. Since the acid exchange processremoves water from the poly alpha-1,3-glucan, introduction ofacid-exchanged poly alpha-1,3-glucan to an esterification reaction withan acid anhydride does not introduce water which may react with the acidanhydride.

Example 2 Preparation of Poly Alpha-1,3-Glucan Acetate

This Example describes producing the glucan ester derivative, polyalpha-1,3-glucan acetate.

Acid-exchanged poly alpha-1,3-glucan (10 g) as prepared in Example 1using acetic acid was added to a mixture containing 180 mL of aceticacid and 1.84 g of sulfuric acid in a 500-mL round bottom flask equippedwith a magnetic stir bar, thermocouple and condenser. This mixture wasstirred for 1 minute at ambient temperature, afterwhich acetic anhydride(50 mL) was added to the mixture. The reaction was allowed to proceedfor 30 minutes at ambient temperature, and then heated in a water bathat 35° C. for 20 minutes followed by heating to 50° C. for 30 minutes.The resulting reaction preparation did not contain any solids. Thereaction was then removed from the water bath and allowed to chill for15 minutes to reach 42° C. The reaction was then quenched with 25 mL of70% acetic acid and stirred for 40 minutes. Poly alpha-1,3-glucanacetate was precipitated using an air-powered blender and DI water. Thesolid was washed twice with water for 30 minutes, followed by one washwith 5% sodium bicarbonate. The poly alpha-1,3-glucan acetate solid wasthen finally washed with water until neutral pH was achieved (two waterwashes). The solid was collected by vacuum filtration, dried undervacuum, and characterized by NMR and SEC. This method yielded polyalpha-1,3-glucan acetate with a DoS of 2.3 and an M_(n) of 29170.

Thus, the ester derivative, poly alpha-1,3-glucan acetate, was preparedand isolated.

Example 3 Additional Preparation of Poly Alpha-1,3-Glucan Acetate

This Example describes producing the glucan ester derivative, polyalpha-1,3-glucan acetate, using various reaction conditions.

Acid-exchanged poly alpha-1,3-glucan was prepared as in Example 1 usingacetic acid. A mixture of 180 mL acetic acid and 0.08 g of concentratedsulfuric acid was prepared in a 500-mL round bottom flask equipped witha magnetic stir bar and thermocouple; this mixture was chilled to 18° C.The acid-exchanged poly alpha-1,3-glucan (10 g) was slowly added to thechilled mixture and stirred for 1 minute. Acetic anhydride (50 mL) wasthen added to the mixture. The reaction was allowed to proceed for 10minutes with no heating, and then heated in a water bath at 35° C. for20 minutes. The resulting reaction, which was devoid of any solid, waschilled to 22° C. over 7 minutes using an ice bath. The reaction wasthen quenched with 25 mL of 70% acetic acid and stirred for 40 minutes.Poly alpha-1,3-glucan acetate was precipitated, washed and analyzed asdescribed in Example 2. This process yielded poly alpha-1,3-glucanacetate with a DoS of 2.41 and an M_(n) of 73960.

Using different concentrations of reagents allowed for different esterproducts to be formed. Table 1 below shows different polyalpha-1,3-glucan acetate esters synthesized using processes similar tothe above process, but with certain modifications as indicated in thetable. The results in Table 1 indicate that by altering the reactionconditions and the molecular weight of poly alpha-1,3-glucan startingmaterial used in the reaction, the DoS by acetyl groups in the esterproduct, as well as the molecular weight of the product, can be altered.

TABLE 1 Poly Alpha-1,3-Glucan Acetate Prepared from PolyAlpha-1,3-Glucan Amount of acetic anhydride Poly alpha-1,3- Reaction andsulfuric acid catalyst Poly alpha-1,3- glucan starting time and used ineach reaction glucan acetate material temp^(a). Acetic Sulfuric productM_(n) Amount (g) ° C. min anhydride (mL) Acid (%) M_(n) DoS 66K 10 36 4050 0.9 73960 2.41 66K 10 30 45 50 1.6 78160 2.55 78K 50 47 35 84 1.858800 2.57 66K 10 36 30 50 0.8 48940 2.15 66K 10 32 45 50 0.8 87510 2.6^(a)Temperature after heating the reaction.

Thus, various forms of the ester derivative, poly alpha-1,3-glucanacetate, were prepared and isolated.

Example 4 Additional Preparation of Poly Alpha-1,3-Glucan Acetate

This Example describes a process having a hydrolysis step for producingpoly alpha-1,3-glucan acetate.

Acid-exchanged poly alpha-1,3-glucan (28 g) as prepared in Example 1using acetic acid was added to a mixture containing 93.4 mL of aceticacid and 2.24 g of concentrated sulfuric acid and mixed. This mixturewas added to a 1-L jacketed reaction vessel equipped with an overheadstirrer and thermocouple, and chilled to 12° C. using a recirculatingbath. The reaction mixture was then stirred for 1 minute before aceticanhydride (89 mL) was added. The reaction was heated using arecirculation bath set at 42° C. for 40 minutes. The reaction at thisstage, which was devoid of any solid, was quenched with 15.25 g (24%)magnesium acetate with excess water to reduce sulfuric acid content to2%. The reaction was then heated to 100° C. over 25 minutes, afterwhichit was stirred at this temperature for 2 hour. The reaction wascompletely quenched by adding 24% magnesium acetate in 5% excess (6.1g). Poly alpha-1,3-glucan acetate was precipitated, washed and analyzedas in Example 2. This process yielded poly alpha-1,3-glucan acetate witha DoS of 2.58.

Thus, the ester derivative, poly alpha-1,3-glucan acetate, was preparedusing a method incorporating a hydrolysis step.

Example 5 Preparation of Poly Alpha-1,3-Glucan Acetate Via Hydrolysis ofPoly Alpha-1,3-Glucan Triacetate

This Example describes, in part, preparing poly alpha-1,3-glucan acetateby hydrolysis of poly alpha-1,3-glucan triacetate.

Poly alpha-1,3-glucan triacetate was first prepared as follows.

Acetic acid (384 mL), acetic anhydride (990 mL), and methylene chloride(890 mL) were mixed. This preparation was added to a 4-L glass reactionvessel equipped with an overhead stirrer and thermocouple, and chilledto 12° C. Acid-exchanged poly alpha-1,3-glucan (130 g) as prepared inExample 1 using acetic acid was slowly added to the chilled mixture andstirred for 1 minute. Perchloric acid (0.1 N) in acetic acid (180 mL)was then added. The reaction was allowed to proceed at ambienttemperature for 3 hour and 35 minutes. The reaction, which was devoid ofany solids, was then added to an air-powered blender containing methanolto precipitate poly alpha-1,3-glucan triacetate. The polyalpha-1,3-glucan triacetate solid thus formed was washed for 30 minuteswith methanol followed by two washes with deionized (DI) water and onewash with 5% sodium bicarbonate. The poly alpha-1,3-glucan triacetatesolid was then finally washed with water until neutral pH was achieved(two water washes) and collected by vacuum filtration, dried undervacuum, and characterized by NMR and SEC. The poly alpha-1,3-glucantriacetate produced had a DoS of 3.0 and an M_(n) of 132300.

The poly alpha-1,3-glucan triacetate produced above was set up forhydrolysis by first dissolving it in 80 mL of acetic acid. DI water (4mL) was then added to this preparation and stirred using a magnetic stirbar until thoroughly mixed. The preparation was then transferred to aParr Reactor (Parr Instrument Company, Moline, Ill.); steam at apressure of 5 kg/cm² was blown into the reactor to raise the temperatureto 150° C. in 12 minutes. The preparation was held at this temperaturefor 50 minutes. The pressure was then increased from 5 kg/cm² to 8.37kg/cm², afterwhich the reaction vessel was chilled to ambienttemperature. The preparation retrieved from the reactor was yellow incolor. However, upon addition of DI water, white solids of polyalpha-1,3-glucan acetate precipitated. This poly alpha-1,3-glucanacetate was isolated using vacuum filtration, and washed and analyzed asin Example 2. This process yielded poly alpha-1,3-glucan acetate with aDoS of 2.4 and an M_(n) of 44200.

Thus, poly alpha-1,3-glucan acetate with a DoS below 2.75 was preparedfrom poly alpha-1,3-glucan triacetate.

Example 6 Preparation of Poly Alpha-1,3-Glucan Propionate

This Example describes producing the glucan ester derivative, polyalpha-1,3-glucan propionate.

Acid-exchanged poly alpha-1,3-glucan (M_(n) of 119130) was prepared asdescribed in Example 1 except that propionic acid was used instead ofacetic acid. Propionic acid (8 mL) and sulfuric acid (0.03 g) were mixedin a 250-mL round bottom flask and chilled to 18° C. The acid-exchangedpoly alpha-1,3-glucan (2 g) was slowly added to the chilled mixture andstirred for 1 minute. Propionic anhydride (10 mL) was then added to thispreparation, afterwhich 0.6 mL glacial acetic acid was added. Thereaction was allowed to proceed for 5 minutes with no heating, and thenheated in a water bath at 42° C. for 1 hour and 45 minutes. The maximumtemperature was not allowed to go beyond 43° C. to avoid excessdegradation of molecular weight. The resulting reaction preparation,which was devoid of any solids, was chilled to 20° C. using an ice bathover 5 minutes. The reaction was then quenched with 4 mL of 50% aqueousacetic acid and stirred for 45 minutes. Poly alpha-1,3-glucan propionatewas precipitated using an air-powered blender and DI water. The solidwas washed twice with water for 30 minutes followed by a wash with 5%sodium bicarbonate. The poly alpha-1,3-glucan propionate solid was thenwashed with water until neutral pH was achieved (two water washes). Thesolid was collected by vacuum filtration, dried under vacuum, andcharacterized by NMR and SEC. The solid was confirmed as polyalpha-1,3-glucan propionate with 44.1 wt % propionyl groups (0 wt %acetyl groups) and an M_(n) of 59510.

Thus, the ester derivative, poly alpha-1,3-glucan propionate, wasprepared and isolated.

Example 7 Preparation of Poly Alpha-1,3-Glucan Acetate Butyrate

This Example describes producing the glucan mixed ester derivative, polyalpha-1,3-glucan acetate butyrate.

Acid-exchanged poly alpha-1,3-glucan (10 g) as prepared in Example 1with acetic acid was added to a mixture containing 21 mL of glacialacetic acid, 20 mL of butyric acid and 0.09 g of sulfuric acid in a500-mL round bottom flask equipped with a magnetic stir bar,thermocouple and condenser. The mixture was chilled to 18° C. using anice bath and stirred for 1 minute before butyric anhydride (39 mL) wasadded to the flask. The reaction was allowed to proceed for 10 minuteswith no heating, and then heated in a water bath at 35° C. for 80minutes, followed by heating to 39° C. for 30 minutes where the maximumtemperature reached was 39° C. to avoid excess degradation of productmolecular weight. The resulting viscous solution, which was devoid ofany solids, was cooled to 20° C. using an ice bath for 10 minutes. Thereaction was then quenched with 20 mL of 50% aqueous acetic acid andstirred for 40 minutes. Solid poly alpha-1,3-glucan acetate butyrate wasprecipitated using an air-powered blender and DI water. The solid waswashed twice with water for 30 minutes, followed by washing with 5%sodium bicarbonate. The solid thus obtained was then finally washed withDI water until neutral pH was achieved (two water washes). The solid wascollected by vacuum filtration, dried under vacuum, and characterized byNMR and SEC. This process yielded poly alpha-1,3-glucan acetate butyratemixed ester with a butyryl DoS of 1.0, an acetyl DoS of 1.3, and anumber-average molecular weight (M_(n)) of 66340.

Using different concentrations of reagents allowed for different mixedester products to be formed. Table 2 below shows different polyalpha-1,3-glucan acetate butyrate esters synthesized using processessimilar to the above process, but with certain modifications asindicated in the table. The results in Table 2 indicate that by alteringthe reaction conditions and the molecular weight of polyalpha-1,3-glucan starting material used in the reaction, the amount ofacetyl and butyryl groups in the mixed ester product, as well as themolecular weight of the product, can be altered.

TABLE 2 Poly Alpha-1,3-Glucan Acetate Butyrate Prepared from PolyAlpha-1,3-Glucan Poly alpha-1,3- Amount of acetic acid, butyric acid,butyric anhydride, Reaction Poly alpha-1,3-glucan glucan starting aceticanhydride and sulfuric acid used in each reaction time and acetatebutyrate product material Acid Acetic Butyric Butyric Acetic Sulfurictemp^(b). wt % wt % M_(n) Amount (g) exchange^(a) acid (mL) Acid (mL)Anhydride (mL) Anhydride (mL) Acid (g) min ° C. M_(n) acetyl butyryl62714 10 acetic 5 35 50 0 0.09 100 36 58680 12.8 32.6 47009 2 butyric 18 10 0 0.03 97 48 67700 1.8 42.7 47009 2 butyric 1 8 10 0 0.03 70 5029777 2.7 37.8 119130 2 acetic 4 4 3 7 0.02 54 40 145300 34.7 9.4^(a)Acid exchange performed following procedure of Example using eitheracetic acid or butyric acid. ^(b)Temperature after heating the reaction.

Thus, various forms of the mixed ester derivative, poly alpha-1,3-glucanacetate butyrate, were prepared and isolated.

Example 8 Preparation of Poly Alpha-1,3-Glucan Acetate Propionate

This Example describes producing the glucan mixed ester derivative, polyalpha-1,3-glucan acetate propionate.

Acid-exchanged poly alpha-1,3-glucan was prepared as described inExample 1 using acetic acid. A mixture of 35 mL of propionic acid and0.09 g sulfuric acid was prepared in a 500-mL round bottom flask andchilled to 18° C. Acid-exchanged poly alpha-1,3-glucan solid (10 g) wasslowly added to the chilled mixture and stirred for 1 minute. Propionicanhydride (50 mL) was then added, afterwhich 5 mL of glacial acetic acidwas added. The reaction was allowed to proceed for 10 minutes with noheating, and then heated in a water bath at 30° C. for 1 hour, followedby heating to 34° C. for 10 minutes. The maximum temperature was notallowed to go beyond 36° C. to avoid excess degradation of productmolecular weight. The solution thus obtained, which was devoid of anysolids, was chilled to 20° C. in an ice bath for 5 minutes. The reactionwas then quenched with 20 mL of 50% aqueous acetic acid and stirred for40 minutes. Poly alpha-1,3-glucan acetate propionate was precipitatedfrom the solution using an air-powered blender and DI water. The solidpoly alpha-1,3-glucan acetate propionate product was washed twice withwater for 30 minutes followed by a wash with 5% sodium bicarbonate. Thesolid was then washed with water until neutral pH was achieved (twowater washes). The solid was collected by vacuum filtration, dried undervacuum, and characterized by NMR and SEC. The solid created wasconfirmed as a poly alpha-1,3-glucan acetate propionate containing 17.6wt % acetyl and 32.9 wt % propionyl groups and having an M_(n) of 64030.

Using different concentrations of reagents allowed for different mixedester products to be formed. Table 3 below shows different polyalpha-1,3-glucan acetate propionate esters synthesized using processessimilar to the above process, but with certain modifications asindicated in the table. The results in Table 3 indicate that by alteringthe reaction conditions and the molecular weight of polyalpha-1,3-glucan starting material used in the reaction, the amount ofacetyl and propionyl groups in the mixed ester product, as well as themolecular weight of the product, can be altered.

TABLE 3 Poly Alpha-1,3-Glucan Acetate Propionate Prepared from PolyAlpha-1,3-Glucan Poly alpha-1,3- Amount of acetic acid, propionic acid,propionic Reaction Poly alpha-1,3-glucan glucan starting anhydride andsulfuric acid used in each reaction time and acetate propionate productmaterial Acid Acetic Propionic Propionic Sulfuric temp^(b). wt % wt %M_(n) Amount (g) exchange^(a) acid (mL) Acid (mL) Anhydride (mL) Acid(g) min ° C. M_(n) acetyl propionyl 62714 10 propionic 3 29 55 0.08 13538 54460 7.4 35.9 62714 10 propionic 5 35 50 0.09 135 38 53450 4.9 41.171127 10 propionic 3 35 50 0.15 75 53 66190 1.7 47.1 47009 2 propionic 18 9 0.03 60 40 61640 6.5 41.6 25587 1 propionic 0.3 3.5 5 0.009 56 4321380 1.4 49.0 119130 2 propionic 0.6 8 10 0.03 55 45 59150 0 44.1^(a)Acid exchange performed following procedure of Example usingpropionic acid instead of acetic acid. ^(b)Temperature after heating thereaction.

Thus, various forms of the mixed ester derivative, poly alpha-1,3-glucanacetate propionate, were prepared and isolated.

Example 9 Preparation of Poly Alpha-1,3-Glucan Triacetate Using SulfuricAcid as Catalyst

This Example describes producing poly alpha-1,3-glucan triacetate usingsulfuric acid as the catalyst in the reaction.

Acid-exchanged poly alpha-1,3-glucan was prepared as described inExample 1 using acetic acid. A mixture of 180 mL acetic acid and 1.84 gof concentrated sulfuric acid, as a catalyst, was prepared in a 500-mLround bottom flask equipped with an overhead stirrer and thermocouple.Acid-exchanged poly alpha-1,3-glucan (10 g) was slowly added to themixture and stirred under nitrogen for 1 minute. This mixture waschilled to about 18° C. using an ice bath. Acetic anhydride (50 mL) wasadded to the reaction, which was then heated to 80° C. over 45 minutesand allowed to react at this temperature for 30 minutes. The reaction,which was devoid of any solids, was chilled to 40° C. using an ice bathover 5 minutes. The reaction was then quenched with 25 mL of 70% aceticacid and stirred for 30 minutes. Poly alpha-1,3-glucan triacetate wasprecipitated using an air-powered blender (Waring, Torrington, Conn.)and DI water. The solid poly alpha-1,3-glucan triacetate product waswashed with water for 30 minutes twice, followed by washing with 5%sodium bicarbonate. The solid was then washed with water until neutralpH was achieved (two water washes). The solid was collected by vacuumfiltration, dried under vacuum, and characterized by NMR and SEC. Thisprocess yielded 7.8 g of poly alpha-1,3-glucan triacetate with a DoS of3.1 and an M_(n) of 5130. The DoS reading over 3.0 likely reflectsintegration variability typical to the NMR measurement process.

Table 4 below shows different molecular weight poly alpha-1,3-glucantriacetate esters synthesized using processes similar to the aboveprocess, but with certain modifications as indicated in the table. Theresults in Table 4 indicate that by altering the reaction conditions andthe molecular weight of poly alpha-1,3-glucan starting material used inthe reaction, the molecular weight of the product can be altered.

TABLE 4 Poly Alpha-1,3-Glucan Triacetate Esters Prepared from PolyAlpha-1,3-Glucan Using Sulfuric Acid Catalyst Poly alpha-1,3- ReactionSulfuric Poly alpha-1,3- glucan starting time and Acid glucan triacetatematerial temp^(a). (wt % of product M_(n) Amount (g) ° C. min glucan)M_(n) DoS 112K  10 43 40 8 68090 2.75 66K 10 59 60 8 29380 2.94 66K 2839 55 8 n/a 2.91 ^(a)Maximum temperature after heating the reaction.

Thus, various forms of poly alpha-1,3-glucan triacetate were preparedand isolated in reactions using sulfuric acid as a catalyst.

Example 10 Preparation of Poly Alpha-1,3-Glucan Triacetate UsingPerchloric Acid as Catalyst

This Example describes producing poly alpha-1,3-glucan triacetate usingperchloric acid as the catalyst in the reaction.

Acid-exchanged poly alpha-1,3-glucan was prepared as described inExample 1 using acetic acid. A mixture of 384 mL acetic acid, 990 mLacetic anhydride, and 890 mL methylene chloride was prepared in a 4-Lglass reaction vessel equipped with an overhead stirrer andthermocouple, and chilled to 12° C. Acid-exchanged poly alpha-1,3-glucan(130 g) was slowly added to the chilled mixture and stirred for 1minute. Perchloric acid (0.1 N) in acetic acid (180 mL) was then addedto the mixture. The reaction was allowed to proceed at ambienttemperature for 3 hour and 35 minutes. The resulting reaction, which wasdevoid of solid, was added to an air-powered blender containing methanolto precipitate poly alpha-1,3-glucan triacetate. The polyalpha-1,3-glucan triacetate solid was washed for 30 minutes withmethanol followed by washing twice with DI water and one wash with 5%sodium bicarbonate. The poly alpha-1,3-glucan triacetate was then washedwith water until neutral pH was achieved (two water washes). The solidwas collected by vacuum filtration, dried under vacuum, andcharacterized by NMR and SEC. This process produced 221.5 g of polyalpha-1,3-glucan triacetate with a DoS of 3.2 and an M_(n) of 132300.The DoS reading over 3.0 likely reflects integration variability typicalto the NMR measurement process.

Table 5 below shows different molecular weight poly alpha-1,3-glucantriacetate esters synthesized using processes similar to the aboveprocess, but with certain modifications as indicated in the table. Theresults in Table 5 indicate that by altering the reaction conditions andthe molecular weight of poly alpha-1,3-glucan starting material used inthe reaction, the molecular weight of the product can be altered.

TABLE 5 Poly Alpha-1,3-Glucan Triacetate Esters Prepared from PolyAlpha-1,3-Glucan Using Perchloric Acid Catalyst Poly alpha-1,3- ReactionPoly alpha-1,3- glucan starting time and glucan triacetate materialtemp^(a). Perchloric product M_(n) Amount (g) ° C. min Acid (g) M_(n)DoS 126K  130 32 180 1.80 132300 3.0 82K 30 30 270 0.49 130700 3.0 66K130 41 150 2.17 93510 2.75 98K 130 37 215 1.80 197000 3.0 ^(a)Maximumtemperature after heating the reaction.

Thus, various forms of poly alpha-1,3-glucan triacetate were preparedand isolated in reactions using perchloric acid as a catalyst.

Example 11 Preparation of Films Using Poly Alpha-1,3-Glucan Acetate

Poly alpha-1,3-glucan acetate, prepared as in Example 2, was dissolvedin acetone at 10 wt % mixture to make a solution. The solution was thencast onto a clean glass plate with a film caster and the solvent wasallowed to evaporate to dryness to provide a film. The film was removedfrom the glass and rinsed with DI water. Table 6 shows properties of twodifferent poly alpha-1,3-glucan acetate films prepared using twodifferent samples of poly alpha-1,3-glucan acetate.

TABLE 6 Poly Alpha-1,3-Glucan Acetate Films Poly Poly alpha- alpha-1,3-1,3-glucan glucan Breaking Tear Average acetate acetate Stress (gf/ TearThickness DoS M_(n) (kgf/mm²) mil) (gf) (microns) 2.6 87510 3.97 2.4 5.658.4 2.74 57240 4.81 2.0 6.8 86.4

Example 12 Optical Analysis of Poly Alpha-1,3-Glucan Acetate Films

A sample of poly alpha-1,3-glucan acetate film, prepared as in Example11, was analyzed for color and haze. The spectra that were collectedwere consistent with ASTM E1164-09a. Spectral Bandwidth (SBW)=1, at 1 nminterval and wavelength range=830-360 nm. Table 7 shows the results ofthis study.

TABLE 7 Optical Measurements of a Poly Alpha-1,3-Glucan Acetate FilmPoly alpha- Poly alpha- 1,3-glucan 1,3-glucan acetate acetate DoS M_(n)Haze Transmittance 2.6 78160 6.32% 5.36%

Example 13 Preparation of Poly Alpha-1,3-Glucan Triacetate Films

Poly alpha-1,3-glucan triacetate was prepared as described in Example10. A 10 wt % solution of the poly alpha-1,3-glucan triacetate wasprepared by dissolving 10 g of it in 90 g methylene chloride:methanol(11.5:1 v/v). This solution was then cast onto a clean glass plate witha Gardner Knife (Gardner Lab Inc., Bethesda, Md.). The solvent wasallowed to evaporate to dryness. The film produced following solventevaporation was removed from the glass and rinsed with DI water. Table 8summarizes the tensile and tear data for poly alpha-1,3-glucantriacetate films produced using this method. It can be seen thatvariation of the M_(n) and DoS of the constituent glucan ester resultsin providing different physical properties for the film produced.

TABLE 8 Poly Alpha-1,3-Glucan Triacetate Films Poly Poly alpha-1,3-alpha-1,3- glucan glucan Breaking Tear/ Average triacetate triacetatestress Thickness Tear Thickness DoS M_(n) (kgf/mm²) (gf/mil) (gf)(micrometers) 2.75 93510 4.98 1.4 6.9 129.5 3.0 93670 6.44 2.9 11.6101.6 3.0 132300 5.9 2.5 14.8 152.4 3.0 197000 5.26 3.1 18.1 147.5

Example 14 Thermal Analysis of Poly Alpha-1,3-Glucan Triacetate Films

Poly alpha-1,3-glucan triacetate films as prepared in Example 13 wereanalyzed using MDSC and TGA. MDSC measurements were performed with 5-6mg of film at a heating rate of 3° C./min, modulation amplitude of 0.48°C., and modulation period of 60 seconds starting from 0° C., in N₂ usingQ1000 TA instrument.

TGA experiments were performed from ambient temperature to 800° C. underN₂ using Q500 TA instrument.

The information provided in Table 9 shows the heat stability/thermaldegradation of the poly alpha-1,3-glucan triacetate films preparedaccording to the method disclosed above. Table 9 summarizes dataacquired from the MDSC and TGA measurements. It can be seen thatvariation of the M_(n) and DoS of the constituent glucan ester resultsin providing different physical properties for the film produced.

TABLE 9 MDSC and TGA Data for Poly Alpha-1,3-Glucan Triacetate FilmsPoly alpha- Tm (° C.), Tm (° C.), 1,3-glucan ΔH (J/g) ΔH (J/g) Onset oftriacetate Tg (° C.) Total Non-Rev Heat Decomposition (DoS/M_(n))(Reverse) Heat flow flow (° C.) 2.75/93510 188.0 333.0 332.9 349.3 29.616.8  3.0/197000 189.1 340.5 339.9 356.7 39.4 29.6

Example 15 Optical Analysis of Poly Alpha-1,3-Glucan Triacetate Films

Poly alpha-1,3-glucan triacetate films prepared as in Example 13 wereanalyzed for color and haze. Spectra were collected consistent with ASTME1164-09a using spectral Bandwidth (SBW) of 1, at 1 nm interval andwavelength range of 830-360 nm. Results of optical measurements of polyalpha-1,3-glucan triacetate films are shown in Table 10.

TABLE 10 Optical Measurements of Poly Alpha-1,3-Glucan Triacetate FilmsPoly alpha- Poly alpha- 1,3-glucan 1,3-glucan triacetate triacetate HazeTransmittance DoS M_(n) (%) (%) 3.0 130700 6.32 91.6 2.75 93510 1.4192.1 2.72 53690 1.37 92.3

What is claimed is:
 1. A film comprising poly alpha-1,3-glucan esterhaving at least one of: (a) a tear resistance of at least about 0.1gf/mil; or (b) a haze of less than about 20% wherein the polyalpha-1,3-glucan ester is a mixed ester.
 2. The film of claim 1, whereinthe poly alpha-1,3-glucan ester comprises two or more of acetate,propionyl, butyryl, pentanoyl, hexanoyl, heptanoyl or octanoyl groups.3. The film of claim 1, wherein the film has at least one of: (a) a tearresistance from about 2.0 to about 2.4 gf/mil; or (b) a haze of lessthan about 10%.
 4. The film of claim 3, wherein the polyalpha-1,3-glucan ester comprises two or more of acetate, propionyl,butyryl, pentanoyl, hexanoyl, heptanoyl or octanoyl groups.
 5. A methodto prepare a poly alpha-1,3-glucan ester film comprising: (a) providingpoly alpha-1,3-glucan mixed ester; (b) contacting the polyalpha-1,3-glucan ester of (a) with a solvent to make a solution of thepoly alpha-1,3-glucan mixed ester; (c) applying the solution of the polyalpha-1,3-glucan mixed ester on a surface; and (d) allowing the solventto evaporate to provide the poly alpha-1,3-glucan mixed ester film. 6.The method of claim 5, wherein the poly alpha-1,3-glucan mixed estercomprises two or more of acetate, propionyl, butyryl, pentanoyl,hexanoyl, heptanoyl or octanoyl groups.
 7. The method of claim 5,wherein the solvent is acetone.
 8. The film of claim 1 wherein the mixedester comprises two or more of acetate, propionyl and butyryl groups. 9.A film comprising poly alpha-1,3-glucan ester having at least one of:(a) a tear resistance of at least about 0.1 gf/mil; or (b) a haze ofless than about 20%, wherein the degree of substitution is up to about2.80.